Methods and compositions for extracting proteins from cells

ABSTRACT

The present invention relates to a process of releasing a protein, recombinant or otherwise, from a cell. The process of the present invention involves contacting a host cell containing a protein of interest with a solution comprising one or more detergents and one or more reducing agents. The methods of the invention are particularly suitable to large scale production of recombinant products.

This application is a continuation-in-part of Ser. No. 09/766,043, filedJan. 19, 2001, now abandoned.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a process of recovering intracellularproteins and other molecules from a cell.

BACKGROUND OF THE INVENTION

It is desirable to lyse cells grown as production hosts containing aprotein or other molecule of interest to recover any desiredintracellularly produced product. Conventional ways to kill and lysesuch cells include the use of heat (U.S. Pat. No. 4,601,986 to Wegner,et al.), osmotic pressure (U.S. Pat. No. 4,299,858 to Aubert, et al),enzymes which break down the cell walls or membranes (U.S. Pat. No.3,816,260 to Sugiyama, U.S. Pat. No. 3,890,198 to Kobayashi, et al. andU.S. Pat. No. 3,917,510 to Kitamura, et al.) and mechanical disruptionof the cell wall by, for example, high pressure homogenization. Thedisclosures of the above patents are incorporated herein by reference.

Also, detergents have been utilized to lyse the cell wall. For example,yeast protein extraction reagent (Y-PER®), sold by Pierce ChemicalCompany, contains a detergent to provide a gentle means of cell lysisthat is not detrimental to the protein of interest. However, Y-PER® isintended to be used as a laboratory bench reagent, not as a reagentuseful for the large scale production of proteins, and is costly. Forthese reasons, Y-PER® has not gained acceptance as a useful reagent forthe large scale production of recombinant protein from host cells.

There is a need in the art for a process that can be used to easilycause the release of proteins from host cells without harming thedesired protein and with a minimum of process steps. The method of celllysis should not directly or indirectly lead to denaturation of thedesired product and the method should be consistent with subsequentprocessing requirements and with large scale production.

SUMMARY OF THE INVENTION

The present invention relates to a process of releasing a protein,recombinant or otherwise, from a cell. The process of the presentinvention involves contacting a host cell containing a protein ofinterest with a solution comprising one or more detergents and one ormore reducing agents. The addition of one or more reducing agentsfacilitates the recovery of proteins in their native conformations. Themethods of the invention are particularly suitable to large scaleproduction of recombinant products. The methods of the inventioncomprise four basic steps: adjustment of bulk solution conditions toachieve a permissive environment, contact of cells with a reducing agenteither before, during or after contact of the cells with certaincharge-modified hydrocarbons, and finally clarification of the extractto produce a fraction suitable for formulation or further processing.Typically, the reducing agent and the detergent are added sequentiallyin any order, resulting in the concurrent exposure of the cells to thereducing agent and the detergent.

In a particular embodiment, the one or more detergents are amphipathic,charged amines or amine oxides coupled to hydrocarbon chains of varyinglengths. In a preferred embodiment, the one or more detergents used areselected from the group consisting of, tributylphosphate,dimethyldecylamine, dimethyltridecylamine, dimethylundecylamine,dimethyldidecylamine, dimethytetradecylamine, dimethylhexadecylamine,dimethyldecylamineoxide, dimethylundecylamineoxide,dimethyldidecylamineoxide, dimethytetradecylamineoxide anddimethyltridecylamineoxide. Preferably, the detergent is notdimethyltridecylamine.

Detergents may be used at concentrations ranging from 0.01% up to theirsolubility limit. Preferably, the concentration of the detergents rangesfrom 0.05% to 5%, 0.1% to 2%, or is approximately 0.5% of the totalsolution. When added to cells suspended in buffer, the detergent ispreferably at a higher concentration than the final concentration atwhich the cells are lysed. Preferably, the detergent is at least at a 2fold, 5 fold, 10 fold or 100 fold higher concentration.

In a particular embodiment, the one or more reducing agents are agentsare those reducing agents that reduce disulfide bonds and/or maintainsulfhydryl residues in the reduced form. Any such reducing agent oragents may be used. In a preferred embodiment, the one or more reducingagents used are selected from the group consisting of, Dithiothreitol(DTT); Dithioerythiritol (DTE); Cysteine (Cys) and Tris2-carboxyethyphosphine (TCEP).

Reducing agents may be used at concentrations ranging from 0.1 mM to 100mM, 1 mM to 25 mM, 2 mM to 10 mM, or about 5 mM. When added to cellssuspended in buffer, the reducing agent is preferably at a higherconcentration than the final concentration at which the cells are lysed.Preferably, the detergent is at least at a 2 fold, 5 fold, 10 fold or100 fold higher concentration.

In addition to the one or more detergents and reducing agents, in apreferred embodiment, the cells are also contacted with glycerol.Preferably, the glycerol concentration is at least 0.6%, or ranges from0.6% to 20%, 0.6% to 15%, 0.6% to 12%, 0.6% to 6%, 0.6% to 3%, or 0.6%to 1%.

The pH of the solution can range from pH 2 to pH 12. Preferably, thesolution is at a pH ranging from pH 5.0 up to pH 8.0. More preferably,the pH ranges from pH 5.5 to 7.4, from pH 6 to 7.4, from pH 7.0 to 7.4,or is approximately pH 7.3.

The recovery of protein from the cells with the solution of theinvention can be carried out at a temperature of from about 2° C. toabout 50° C. Preferably, the temperature is from about 2° C. to about30° C., about 2° C. to about 20° C., about 2° C. to about 10° C., about3° C. to about 10° C., about 4° C., about 25° C., or at roomtemperature.

The “host cells” are cells containing a protein of interest. A “proteinof interest” is any protein present in a host cell that one desires torelease from the host cell and, optionally, subsequently isolate orpurify. Preferably, the protein of interest is a recombinant protein. Ina preferred embodiment, the protein of interest has a molecular weightof less than 100 kD. In a further preferred embodiment, the protein ofinterest has a molecular weight of between 5 and 75 kD, preferably about50 kD. The host cells may be of any type, preferably mammalian,bacterial, yeast, fungal, plant, avian, or reptilian. Most preferably,the host cells are yeast cells. In a particular embodiment, the yeastcells are of the species Pichia pastoris.

In addition to releasing proteins from host cells, the composition ofthe invention may be used to release other molecules from host cellsincluding nucleic acids, lipids, vitamins, small molecules and othercell, cytosolic, or organelle derived molecules or molecular complexes.

BRIEF DESCRIPTION OF THE DRAWINGS

The various features of the invention may be more fully understood fromthe following description when read together with the accompanyingdrawings.

FIG. 1 . Extraction of rBoNTA(Hc) with and without DTT at 4° C. and 21°C.—FIG. 1 shows the effect of time, temperature and reducing agent onthe recovery of a protein of interest from a host cell.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a process of releasing a protein,recombinant or otherwise, from a cell. The methods of the inventioncomprise four basic steps: adjustment of bulk solution conditions toachieve a permissive environment, contact of cells with a reducing agenteither before, during or after contact of the cells with certaincharge-modified hydrocarbons, and finally clarification of the extractto produce a fraction suitable for formulation or further processing.

In a particular embodiment, solution conditions are adjusted by 1)sedimentation of the cellular fraction followed by re-suspension, 2) bysolution exchange using filtration methods, 3) by direct modification ofthe existing solution conditions, or 4) by other means of solventexchange. Cells suspended in an appropriate solution are then contactedwith certain amphipathic molecules that cause disruption of the cellmembrane thus allowing the cell contents to diffuse through the cellwall and into the extracellular medium. Cellular debris is thenseparated from the soluble extract by sedimentation, flocculation,filtration, chromatographic, or other separation methods.

In a preferred embodiment, yeast cells, for example Pichia pastoris,taken directly from a fermentation process are concentrated and thenexchanged from growth medium into a specific buffered solution bytangential flow filtration using microporous membranes. The concentratedand solution adjusted cells are then contacted with dimethylamine and/ordimethylamine oxide compounds with alkyl chains of varying length,depending on cell type and solution conditions. In addition, thesolution may contain detergents, such as Triton X-100 or polyols, suchas glycerol, that enhance the extraction process. The extract ofintracellular molecules is then separated from the remaining insolubleslurry by depth filtration, for example, using diatomaceous earth in aplate and frame filter press.

Solution permissivity is dependent on pH, temperature, ionic strength,time, cell concentration and the addition of certain components thatenhance the effectiveness or kinetics of cell disruption or molecularstability. Solution pH can be from pH 3 to 11, more preferably 5 to 8,or approximately 6.5 to 8.0. Temperature can range from about 20° C. to50° C., or more preferably 2° C. to 30° C. Ionic strength can vary fromhypotonic to hypertonic conditions, most preferably 0.001 to 3000 mMsodium chloride, or other salt, depending on the properties of thedesired target molecule. In preferred embodiments, the ionic strength isbelow 350 mM, below 250 mM, below 150 mM, below 100 mM, below 50 mM, andbelow 10 mM, but not lower than 5 mM, 1 mM, 0.01 mM or 0.001 mM.

The “host cells” are cells containing a protein of interest. A “proteinof interest” is any protein present in a host cell that one desires torelease from the host cell and, optionally, subsequently isolate orpurify. Preferably, the protein of interest is a recombinant protein.When the protein of interest is a recombinant protein, it can beproduced by any method known in the art. Typically, a gene that encodesthe recombinant protein that is desired is inserted into a recombinantmolecule. The polynucleotides constituting the gene may be obtained bystandard procedures known in the art, such as from cloned DNA (such as aDNA “library”), chemical synthesis, cDNA cloning, or by the cloning ofgenomic DNA, or fragment thereof, from a desired cell as described inSambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2dEdition, Cold Spring Harbor Laboratory Press (1989).

Once the gene encoding the recombinant protein has been isolated, it isinserted into an appropriate cloning vector. A large number ofvector-host systems known in the art may be used. Possible vectorsinclude, but are not limited to, plasmids or modified viruses, providedthat the vector system is compatible with the host cell used. Thevectors that can be used include, for example, an E. coli cloningvector, bacteriophages such as lambda derivatives, plasmids such aspBR322 derivatives or pUC plasmid derivatives. The cloning vector can beintroduced into host cells via transformation, transfection, infection,electroporation, etc., so that many copies of the gene sequence aregenerated.

Transformation of host cells with a cloning vector that incorporates thegene enables the generation of multiple copies of the gene. Therefore,the gene may be obtained in large quantities by growing transformants,isolating the cloning vector from the transformants and, when needed,retrieving the inserted gene from the isolated cloning vector. Oncesufficient copies of the gene sequence have been generated, the geneencoding the recombinant protein, or a functionally active fragment orother derivative thereof, can be inserted into an appropriaterecombinant molecule. The recombinant molecule is a polynucleotideexpression vector that contains the necessary elements for thetranscription and translation of the inserted protein-coding sequence ofthe recombinant protein. Preferably, the expression vector also includesan origin of replication. The necessary transcription and translationsignals can also be supplied by the native gene and/or its flankingregions.

Once a recombinant molecule has been prepared, it is inserted into anacceptable host cell which will grow and divide to produce clones. Avariety of host cell-vector systems may be utilized to express therecombinant protein. Suitable host cell-vector systems include, forexample, bacterial expression systems, mammalian cell systems infectedwith a virus, such as a vaccinia virus or adenovirus, insect cellsystems infected with a virus such as a baculovirus, microorganisms suchas yeast containing yeast vectors, and bacteria transformed withbacteriophage DNA, plasmid DNA or cosmid DNA.

Recombinant molecules containing the gene of interest can be identifiedby PCR amplification of the desired plasmid DNA or specific mRNA,nucleic acid hybridization, presence or absence of marker gene functionsand expression of the inserted sequences.

The host cells, such as E. coli cells, transformed with a polynucleotidethat encodes a recombinant protein of interest, can be added directly toa reaction vessel. To purify and recover the recombinant protein, thehost cells are concentrated to form a concentrated suspension of wholecells. The host cells can be concentrated by any method known in theart. For example, the host cells may be centrifuged. Centrifugationremoves water from the host cells and concentrates the cells, forming acell paste. Centrifugation also separates the host cells from theculture medium. The host cells may be of any type, preferably mammalian,bacterial, yeast, plant, avian, or reptilian. Most preferably, the hostcells are yeast cells.

Once a concentrated suspension of cells has been obtained, the cells arecontacted with a protein recovery solution. The protein recoverysolution comprises one or more detergents and one or more reducingagents. In a particular embodiment, the one or more detergents areamphipathic, charged amines or amine oxides coupled to hydrocarbonchains of varying lengths. In a preferred embodiment, the one or moredetergents used are selected from the group consisting of,tributylphosphate, dimethyldecylamine, dimethyltridecylamine,dimethylundecylamine, dimethyldidecylamine, dimethytetradecylamine,dimethylhexadecylamine, dimethyldecylamineoxide,dimethylundecylamineoxide, dimethyldidecylamineoxide,dimethytetradecylamineoxide and dimethyltridecylamineoxide. Preferably,the detergent is not dimethyltridecylamine. Detergents may be used atconcentrations ranging from 0.01% up to their solubility limit.Preferably, the concentration of the detergents ranges from 0.05% to 5%,0.1% to 2%, or is approximately 0.5% or 1% of the total solution.Preferably, the detergents, just prior to their addition to the hostcells, are at least 90%, at least 95% or at least 99% pure. In preferredembodiments, the detergents to be added are at least 3 fold, at least 5fold, at least 10 fold, at least 50 fold, at least 100 fold, at least200 fold the final concentration of the detergents after addition anddilution in the cell suspension.

In a particular embodiment, the one or more reducing agents are agentsare those reducing agents that reduce disulfide bonds and/or maintainsulfhydryl residues in the reduced form. Any such reducing agent oragents may be used. In a preferred embodiment, the one or more reducingagents used are selected from the group consisting of, Dithiothreitol(DTT); Dithioerythiritol (DTE); Cysteine (Cys) and Tris2-carboxyethyphosphine (TCEP).

Reducing agents may be used at concentrations ranging from 0.1 mM to 100mM, 1 mM to 25 mM, 2 mM to 10 mM, or about 5 mM. When added to cellssuspended in buffer, the reducing agent is preferably at a higherconcentration than the final concentration at which the cells are lysed.Preferably, the detergent is at least at a 2 fold, 5 fold, fold or 100fold higher concentration. The effectiveness of the reducing agent canbe determined by titration of free sulfhydryls using Ellman's reagent.Preferably, the reducing agent is Dithiothreitol. Preferably, thereducing agent concentration is 5 mM. Preferably, the reducing agent isadded to the extraction buffer just prior to the addition of thedetergent followed by incubation at 4° C. for 12 hours. However, thereducing agent may be added before or after the detergent. Preferably,the reducing agent is added prior to the addition of the detergent. Inanother embodiment, the detergent and reducing agent are added together.In another embodiment, the detergent is added prior to the reducingagent.

In addition to the one or more detergents and reducing agents, in apreferred embodiment, the cells are also contacted with glycerol.Preferably, the glycerol concentration is at least 0.6%, or ranges from0.6% to 20%, 0.6% to 12%, 0.6% to 6%, 0.6% to 3%, or 0.6% to 1%.Preferably, the glycerol, just prior to addition to the host cells, isat least 90%, at least 95% or at least 99% pure. In preferredembodiments, the glycerol to be added is at least 3 fold, at least 5fold, at least 10 fold, at least 50 fold, at least 100 fold, at least166 fold the final concentration of the glycerol after addition anddilution in the cell suspension.

Preferably, the solution is at a pH ranging from pH 5.0 up to pH 8.0.More preferably, the pH ranges from pH 5.5 to 7.4, from pH 6 to 7.4,from pH 7.0 to 7.4, or is approximately pH 7.3.

The recovery of the protein of interest from the cells with the solutionof the invention can be carried out at a temperature of from about 2° C.to about 50° C. Preferably, the temperature is from about 2° C. to about30° C., 2° C. to about 20° C., 2° C. to about 10° C., about 4° C., about25° C., or at room temperature.

The amount of the detergent and reducing agent solution of the inventionused per gram of cells can vary greatly, for example, anywhere from 0.5mL of detergent and reducing agent solution per gram of cells to 20 mLper gram may be used. Preferably, 2.5-5.0 mL detergent and reducingagent solution per gram of cell paste is used.

The amount of time allowed for lysis of the cells after contacting saidcells with the protein recovery solution may be determined by one ofskill in the art. For example, cells may be incubated in the presence ofthe protein recovery solution for 40 minutes up to 72 hours, preferably90 minutes, 150 minutes, 8 hours or 16-30 hours. Shorter and longertimes are also appropriate. In general, the amount of time can beincreased when the concentration of detergent is low and decreased whenthe amount of detergent is high. For example, a protein recoverysolution with a 1% detergent concentration is effective after 40minutes, while a protein recovery solution with a 0.1% detergentconcentration should be incubated for 150 minutes or longer. For optimalrecovery of protein, the exact amount of time necessary can bedetermined by a simple time-course experiment at a given concentrationof detergent, where concentration of the protein of interest released tothe medium is determined over time. After a certain time point, nofurther increase in released protein will be observed. This time pointis the optimal time necessary for lysis of the cells with the chosenconcentration of detergent.

After lysis of the cells, the solution can be centrifuged to collectcellular debris in the pellet, leaving the released protein of interestin the supernatant. The supernatant may be processed according tomethods known to those of skill in the art to further isolate and purifythe protein of interest. The methods utilized to further isolate and/orpurify the protein of interest are highly dependent upon thecharacteristics and properties of the particular protein of interest,and must be determined for each protein. In a particular embodiment, theprotein of interest has reactive sulfhydryls. In a particularembodiment, the protein of interest, in its native conformation, has oneor more disulfide bonds. In a particular embodiment, the protein ofinterest is purified in a reduced form, e.g., with free sulfhydrylgroups. In a particular embodiment, the protein isolated in a reducedform is folded after purification into its native, oxidizedconformation. For methods of folding proteins, see Chaudhuri JB,Refolding recombinant proteins: process strategies and novel approaches.Ann N Y Acad Sci May 1994 2;721:374-85, incorporated herein in itsentirety by reference.

In a preferred embodiment, the method of the invention is applied to alarge scale process of recovering a biomolecule. A large scale processis a process involving large amounts of host cell biomass. In preferredembodiments, the amount of biomass processed in a single batch accordingto the methods of the invention is from 1 kg to 50,000 kg, 1000 kg to20,000 kg, 5000 kg to 10,000 kg, or about 40 kg, 100 kg, 1000 kg or10,000 kg. In another embodiment, the amount of biomass is greater then1 kg, greater than 40 kg, greater than 100 kg, greater than 1000 kg,greater than 5000 kg, greater than 10,000 kg, or greater than 20,000 kg.The host cells are generally in fermentation broth at the beginning ofthe process. The host cells are concentrated in the fermentation broth,by, for example, centrifugation or tangential flow filtration, then thefermentation broth exchanged with 60 mM sodium phosphate, 50 mM NaCl, 5mM EDTA, pH 7.3 (buffer). The exchange can be performed by any meansknown to one of skill in the art, for example, by tangential flowfiltration using 0.45 μm membrane. The concentrated biomass can bemaintained at a constant volume while buffer is added to the biomass ata rate equal to the rate at which liquid was removed from the biomass bythe filtration process. A buffer exchange of greater than 50%, greaterthan 60%, greater than 70%, greater than 80%, greater than 90%, 95%,greater than 95% or approximately 100% may be used. Preferably, theexchange is 90% or more. For example, for 7.6 liters of fermentationbroth containing approximately 3 kg of cells, approximately 27 L ofbuffer may be processed as above which results in a buffer exchangeof >95%. The concentrated and buffer exchanged biomass is then modifiedby the addition of reducing agent, detergent and other compounds, forexample, glycerol and tetradecyldimethylamine to concentrations of 5 mM,6.0% final weight and 0.5% final weight, respectively. The addedcompounds may be added separately or mixed and added together. Otherconcentrations, as recited above, may also be used. The mixture may thenbe incubated with agitation. This incubation may be for any appropriatetime and temperature, for example, for 14.5 h at 19° C. The actualconcentrations of reducing agents, detergents, time and temperature forany given extraction may be readily determined by one of skill in theart. After incubation, the soluble fraction of the mixture containslarge amounts of protein, nucleic acids, lipids and other moleculespreviously restricted to the cell membrane and cytoplasm. The mixturemay be centrifuged, for example at 4000×g for 30 minutes. Thesupernatant fraction may then be further processed by, for example,filtration through a 1.2 μm and 0.2 μm filters in series.

The following Examples illustrate the preferred embodiments of theprocess of the present invention and is not limiting of thespecification and claims in any way.

EXAMPLES

Large Scale Release of Recombinant Product

Pichia pastoris fermentation broth (12.6 Kg) containing 24.4% biomass bywet weight, having a conductance of 30.2 mS/cm and pH of 4.86, wasconcentrated to 7.6 L. The concentrated biomass was then exchanged into60 mM sodium phosphate, 50 mM NaCl, 5 mM EDTA, pH 7.3 (buffer) bytangential flow filtration using 0.45 m² of 0.45 μm membrane. Theconcentrated biomass was maintained at 7.6 L while buffer was added tothe biomass at a rate equal to the rate at which liquid was removed fromthe biomass by the filtration process. Approximately 27 L of buffer wereprocessed as above which results in a buffer exchange of >95%. Theconcentrated and buffer exchanged biomass was then modified by theaddition of glycerol and tetradecyldimethylamine, separately, toconcentrations of 6.0 and 0.5 final weight percent, respectively. Themixture was then incubated with agitation for 14.5 h at 19 ° C. At thispoint the soluble fraction of the mixture contained large amounts ofprotein, nucleic acids, lipids and other molecules previously restrictedto the cell membrane and cytoplasm. The mixture was then centrifuged at4000×g for 30 minutes. The supernatant fraction was then furtherprocessed by filtration through a 1.2 μm and 0.2 μm filters in series.The resulting clarified extract contained approximately 15 g/L of totalprotein and 0.8 g/L of the specific heterologous protein of interest.The clarified extract also contained significant quantities ofribonucleic acid, deoxyribonucleic acid and cell derived lipids.

Extraction of Recombinant Product Over Time and at DifferingTemperatures

Pichia pastoris cells were exchanged into permissive buffer conditionsand contacted with 0.5% tetradecyldimethylamine (DMA-C14) and 1% TritonX-100 and 6% glycerol. Samples of the supernatant were taken at varioustimes after incubation at different temperatures, 4° C. and 22° C. Theconcentration of a specific heterologous (Bot B, or rBoNTB/Hc protein)protein was then determined by an HPLC method specific for that protein.The results are shown in FIG. 1 (4° C.) and 2 (21° C.). The heterologousis released at higher efficiency over time and at the highertemperature, although at 25 hours, the amount of protein released at thetwo temperatures were indistinguishable when DMA C14(tetradecyldimethylamine), triton and glycerol or DMA-C14 and glycerolwere used. DMA C14 and Triton and DMA C14 were less effective. Otherobservations, not represented in FIG. 1 or FIG. 2, show that glycerol,Triton X-100 or buffer alone did not extract protein at a level that wasdetectable.

Non-Selective Recovery of Intracellular Biomolecules

Pichia pastoris cells were exchanged into permissive buffer conditionsand contacted with 0.5% tetradecyldimethylamine (DMA-C14) and 6%glycerol. Samples were taken after incubation and after clarification(low speed centrifugation and 0.2 μm filtration). The concentration ofRNA, DNA, phospholipids, and total protein were measured usingcommercially available reagent kits. The concentration of a specificheterologous protein (rBoNTB/Hc) of interest was then determined HPLC.The results are in Table 1.

TABLE 1 Phospho- Total heterologous Sample/ RNA DNA lipids proteinprotein assay (mg/L) (mg/L) (mg/L) (g/L) (mg/L) extract 479 61 1000 7.5ND clarified <8 ug/mL 4 <50 ug/mL 6.7 0.36 extract

The data show that RNA, DNA, phospholipids, a specific heterologousprotein, and non-specific total protein were extracted from the cells.This shows that the method is non-selective and therefore generallyapplicable for the recovery in intracellular molecules from microbialcells. The data also show that DNA, RNA, and lipids are easily removedfrom the soluble protein fraction of the DMA-C14 glycerol extract(compare the extract to the clarified extract). This is in contrast tomechanical cell extraction methods that result in the formation ofcolloidal suspensions high in nucleic acid and lipid content. Suchcolloidal materials are not easily separated from the soluble proteinfraction and may complicate protein purification methods. Or, on theother hand, severely limit the recovery of DNA, RNA, lipids or similarmolecules if those molecules were the molecules of interest. Thus, thegentle, low shear and non-selective feature of the permeabilizationmethod may facilitate the recovery of non-protein intracellular productssuch as nucleic acids, lipids, and other molecules not readily obtainedfrom mechanical lysates.

Recovery of Intracellular Biomolecules in the Presence of a ReducingAgent

P. pastoris cells expressing the rBoNTA(Hc) protein were suspended (350g biomass/L extraction buffer) in a solution containing 0.5% DMA-C14(w/w) and 6% (w/w) glycerol at pH 7.2. One sample was then incubated at21° C. and a second sample at 4° C. Dithiothreitol (DTT) was then addedto a third sample to 4 mM and the sample was incubated at 21° C. DTT wasadded to a fourth sample to 1 mM and the sample was incubated at 4° C.Aliquots were removed from each of the four samples at the time-pointsindicated (see FIG. 1), the samples were clarified and the concentrationof rBoNTA(Hc) was determined by HPLC. The rBoNTA(Hc) concentration wasthen plotted as a function of time for the four different treatments.The data are summarized in FIG. 1.

Although the invention is described in detail with reference to specificembodiments thereof, it will be understood that variations which arefunctionally equivalent are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. A method of releasing a protein of interest fromhost cells comprising contacting the host cells with a detergent,wherein the detergent comprises an amphipathic charged amine or anamphipathic charged amine oxide.
 2. The method of claim 1, wherein thedetergent is selected from the group consisting of: dimethyldecylamine,dimethyltridecylamine, dimethylundecylamine, dimethyldidecylamine,dimethyltetradecylamine, dimethylhexadecylamine,dimethyldecylamineoxide, dimethylundecylamineoxide,dimethyldidecylamineoxide, dimethytetradecylamineoxide anddimethyltridecylamineoxide.
 3. The method of claim 1, wherein thedetergent is not dimethyltridecylamine.
 4. The method of claim 1,wherein the host cells are Pichia pastoris cells.
 5. The method of claim1, further comprising the step of incubating the host cells at atemperature of between about 2° C. and about 50° C.
 6. The method ofclaim 1 wherein the detergent is added to the host cells at aconcentration up to two hundred times the concentration of the cells. 7.The method of claim 1 wherein the pH of the host cells ranges from aboutpH 5.0 to about pH 8.0.
 8. The method of claim 1 wherein the host cellsare suspended.
 9. The method of claim 1, further comprising the step ofincubating the host cells from about 40 minutes to about 72 hours. 10.The method of claim 9, wherein the incubation is from about 90 minutesto about 24 hours.
 11. The method of claim 1 further comprisingcontacting the host cells with a reducing agent.
 12. The method of claim11, wherein the reducing agent is selected from the group consisting ofDithiothreitol (DTT); Dithioerythiritol (DTE); Cysteine (Cys) and Tris2-carboxyethyphosphine (TCEP).
 13. The method of claim 12, wherein thereducing agent is at a concentration of from about 0.1 mM to about 100mM.
 14. The method of claim 1, further comprising adding glycerol to thehost cells.
 15. The method of claim 14, wherein the glycerol comprises aconcentration of between about 0.6 to about 20 percent.
 16. The methodof claim 14, wherein the detergent comprises a concentration of betweenabout 0.01 percent to the solubility limit of the detergent.
 17. Themethod of claim 16, wherein glycerol comprises a concentration ofbetween about 0.6 to about 6 percent.